In the expending field of proteomics, complex biological samples that can contain up to 30,000 different proteins have to be separated and analyzed.
Up to now, two-dimensional (2D)-gel electrophoresis has been the technique of choice to identify and classify proteins according to their isoelectric point (pI) and mass (Wilkins, M. R. et al., Proteome Research: New Frontiers in Functional Genomics; Springer, 1997). Indeed, with 2D-gel electrophoresis, proteins are separated first by an isoelectric focusing (IEF) step according to their pI. Secondly, proteins are separated as a function of their molecular mass by a polyacrylamide gel electrophoresis (PAGE) step. The proteins can thus be classified in databases according to their pI and mass.
However, despite its extraordinary resolving power, it appears that 2D-gel electrophoresis has reached a plateau in the number of proteins that can be separated and detected in a single map. Moreover, with 2D-gel electrophoresis, only protein separation and classification according to pI and mass can be achieved. Further analysis of the proteins is required in order to obtain more specific information, such as for example peptide composition or biological activity. For this purpose, the proteins have to be extracted out of the gel matrix and analyzed with the appropriate technique. Extraction procedures such as blotting or spot cutting are time consuming and/or risky for protein recovery and activity.
Once a protein has been extracted, the most powerful analytical technique is mass spectrometry (MS), which allows one to analyze peptide composition. However, for MS analysis, the purity of the samples is critical and they generally have to be treated before measurements, which complicates the procedure. Indeed, if the sample contains salts or other impurities, disadvantages can arise for MS analysis. Thus, a sample with a high concentration of salt needs to be desalted by means such as a dialysis procedure before MS analysis. Moreover, the extraction method is not favorable for MS analysis since it leads to a high concentration of undesired compounds in the recovered sample. Some methods to avoid undesired compounds in the samples have been developed. For example, a direct laser desorption technique from the 2D-gel was described by Ogorzalek Loo R. R. et al. (Analytical Chemistry, 1996, 68, 1910-1917) and an electroblotted 2D-gel was developed by Eckerskorn C. et al. (Analytical Chemistry, 1997, 69, 2888-2892). So the major problem with 2D gel-electrophoresis is that the compounds to be analyzed are trapped within a gel and must be extracted and purified before further analyses.
Another efficient technique to separate complex biological samples is isoelectric separation as for example isoelectric focusing (IEF). There are two major types of IEF devices: free flowing buffered systems and immobilized buffered systems.
The free flowing systems (Soulet, N. et al., Electrophoresis, 1998, 19, 1294-1299; Fuh, C. B. and Giddings, J. C., Separation Science and Technology, 1997, 32, 2945-2967, Bier, U.S. Pat. No. 5,540,826) are based on the use of a buffer (carrier ampholytes or isoelectric buffers). In this case, the major disadvantage is that the final separated fractions contain a certain amount of undesired buffering species or ampholytes. This is a problem since these additional compounds have to be removed before further analyses.
In the case of the immobilized buffering systems, the major disadvantage is that the final separated fractions are trapped in a gel or a membrane, which complicates further analyses. However, a “segmented immobilized pH gradients” device based on the use of multiple compartments separated by immobiline isoelectric membranes was developed by Righetti et al. (Journal of Chromatography, 1989, 475, 293-309). The advantage of this technique is that proteins are recovered in an ampholyte-free solution. However, this method requires the use of multiple compartments, multiple immobilized membranes and segmented pH gradients.
Therefore, the development of new high throughput techniques that allow the separation of complex biological samples and the direct recovery in solution of the compounds to analyze is required. These techniques should minimize separation times, be easy to use and result in a high degree of purity.
Recently, a novel method and apparatus was developed and described by Ros et al. (WO 01/86279). In this case, it is possible to purify compounds that are globally neutral from charged species in a sample which does not need to be buffered. The principle of this method is to flow proteins under an immobilized pH gradient (IPG) gel through which an electric field is applied perpendicular to the direction of the flow. Thanks to the buffering capacity of the IPG gel, proteins in solution close to the gel are extracted with respect to their isoelectric point. Only the proteins with a pI close to the pH of the IPG gel stay in the flowing solution. This technique can be used as a prefractionation step before further analysis, and can also be used as a gel loading method. This technique provides a very high separation rate since the molecules migrate in solution and not in a gel as in IEF systems. The device developed for this OffGel®) electrophoresis is made of:                a chamber above which an immobilized chemical buffering system (e.g. an IPG gl) is inserted, to close the chamber and buffer it at the desired pH range.        two platinum electrodes (placed at each extremity of the device) for producing an electrical current along the IPG gel, above which apertures are made to serve as cathodic and respectively anodic reservoirs and to let the possible gas produced during the purification escape out of the device, and        optionally, an inlet and an outlet, connected via tubing to a peristaltic pump to recirculate the sample.        
In this case, the potential difference is only applied through at least a portion of the chemical buffering system. This electrophoresis technique offers extraordinary potentialities and allows the direct recovery of the compounds of interest in solution. However, the device is not very convenient for modem life sciences such as proteomics, which requires high throughput techniques, with short purification time and optimized compound recovery. Furthermore, the strong drawback of Off-Gel electrophoresis is that only components of pI=pH of the gel in contact with the chamber are recovered in solution. It is indeed clear that any other proteins with pI close to the pH of the gel will migrate inside the gel and will be lost for further analysis. Furthermore, preconcentration of sample is probably difficult to realize with Off-Gel electrophoresis, which is one of the bottlenecks of proteomics for analysis of low abundant proteins.
Various systems have been developed for electrophoretic separation purposes. Even though many of them do use either a multi-compartment device or a gel- or membrane-based support (as e.g. the apparatuses described in EP 0776700, GB1422118, EP 0173081, U.S. Pat. No. 5149418 or U.S. Pat. No. 3719580), they refer to conventional electrophoresis separation systems. Indeed, these systems are based on the classical principles of electrophoretic migration of molecules in an electrical field, and they are generally devoted to the separation of molecules according to their size or mass as as widely used for the separation of DNA molecules or fragments. Indeed, such compounds do not have amphoteric properties (in contrast to e.g. proteins) and hence they always exhibit the same charge during the separation. These electrophoretic separation systems therefore do not allow one to separate compounds according to their respective isoelectric point. Indeed, the gels or membranes used in these systems do not comprise any buffering means to fix the pH, and these electrophoretic separation systems are thus fundamentally different from the aforementioned IEF apparatuses, since they are based on totally different separation principles.